Real-time in vivo visualization of oxidative stress in ...

DOI: 10.2478/s11535-007-0022-1 Research article CEJB 2(3) 2007 351–363

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.) Jaka Razinger∗, Luka Drinovec, Alexis Zrimec Institute of Physical Biology, SI-1290 Grosuplje, Slovenia

Received 20 February 2007; accepted 25 May 2007 Abstract: An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 µM), menadione (50 µM) + ascorbate (100 µM) or neither for control. Menadione (50 µM) caused a statistically significant increase in H2 DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 µM) in the test medium significantly decreased apoplastic oxidative stress. 50 µM menadione caused an increase in symplastic H2 DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved. Keywords: EMCCD camera, in vivo, Lemna minor, non-invasive, oxidative stress, plants, reactive oxygen species, real-time visualization

Abbreviations AAPH, 2,2’-Azobis(2-methyl-propionamidine)-dihydrochloride; A.U., arbitrary unit; EMCCD, electron multiplying charge coupled device; H2 DCFDA, dichlorofluorescein diacetate; H2 DCF, dichlorofluorescin; DCF, dichlorofluorescein; menadione, 2-methyl-1,4-naphtoquinone; PAR, photosynthetic active radiation; ROS, reactive oxygen species ∗

E-mail: [email protected]

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J. Razinger et al. / Central European Journal of Biology 2(3) 2007 351–363

Introduction

Presently, oxidative stress is one of the more emphasized areas of research worldwide. Much research has been done in the past to quantify oxidative stress by various biochemical methods. Those included measurement of antioxidative enzymes (superoxide dismutase, glutathione reductase, catalase, etc.) or non-enzymatic antioxidants (glutathione, ascorbate, uric acid, etc.) or measurement of cellular damage (peroxidation of membrane lipids, nucleic acid or protein degradation, photosynthetic pigment loss, etc.). Less research has been carried out in the field of reactive oxygen species (ROS) visualization. Due to the extremely short life-span of ROS [1] and efficient cellular mechanisms that quench and detoxify them, it is difficult to directly visualize ROS. Therefore, detection techniques have to be very sensitive and fast. Additionally, problems with artefacts occur when one tries to insert a marker into the cell, as this is the usual way to measure actual intracellular oxidation processes [2]. Researchers also emphasize the importance of noninvasive techniques of visualization, if possible on whole organisms, as this is the only way to avoid damaging the test subject and generating artefacts [3, 4]. Interestingly, among the various plant visualization methods and markers used, fluorescent fluorochromes are relatively rarely employed [5]. Dichlorofluorescin (H2 DCF) is a fluorescent marker widely used for determination of H2 O2 [6, 7], and for studies of oxidative stress at the cellular level [8, 9]. In cellular studies, dichlorofluorescein (H2 DCF) is given as a diacetate ester (dichlorofluorescein diacetate; H2 DCFDA), which permeates the plasma membrane and is hydrolyzed inside the cells by unspecific esterases yielding polar products, supposedly trapped within the cell. Non-fluorescent H2 DCF is oxidized to fluorescent dichlorofluorescein (DCF), which is determined in flow cytometers, in fluorimeters after cell disruption or with fluorescent microscopes [8, 10]. A problem that occurs with intracellularly formed H2 DCF is that it does not necessarily remain within the cytoplasm but rather can leak to extracellular media [11], where it would be available for reaction with extracellular oxidants. Also, because of the non specificity of DCF, this marker is best applied as a qualitative marker of cellular oxidative stress rather than a precise indicator of H2 O2 [2]. We report a method for real-time in vivo ROS visualization on a whole organism level with a cooled electron multiplying (EMCCD) camera. The use of an ultra-sensitive EMCCD camera allowed us to use low level excitation light which did not induce significant photobleaching of the fluorescent probe. The test organism in the study was Lemna minor L. (common duckweed), a free-floating monocot macrophyte commonly used in laboratory studies [12]. It consists of photosynthetically active fronds and root-like structures, which also contain chloroplasts. An additional benefit of using plants in this non-invasive technique is their inherent trait of containing esterases in the apoplast [13–16]. This enabled us to observe either apoplastic or symplastic ROS formation in the test plants after exposure to 2-methyl-1,4-naphtoquinone (menadione), a commonly used ROS inducer [8, 17–22], with slight modifications in the fluorescent dye loading protocol.


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Experimental Procedures

2.1 Plant material and growth conditions Lemna minor L. (common duckweed) plants were collected in a local stream and cultured under controlled conditions. They were rinsed with 0.01 M NaOCl for 30 sec to prevent algal outgrowth. A stock culture was grown in 3 l glass containers in a modified Steinberg growth medium containing 3.46 mM KNO3 , 1.25 mM Ca(NO3 )2 · 4H2 O, 0.66 mM KH3 PO4 , 0.072 mM K2 HPO4 , 0.41 mM MgSO4 · 7H2 O, 1.94 µM H3 BO3 , 0.63 µM ZnSO4 · 7H2 O, 0.18 µM Na2 MoO4 · 2H2 O, 0.91 µM MnCl2 · 4H2 O, 2.81 µM FeCl3 · 6H2 O and 4.03 µM EDTA (all chemicals from Fluka, except EDTA from Sigma). The stock culture was kept in growth chambers at 25 ± 1 ◦ C under cool white fluorescent light (160 µM m−2 s−1 PAR) with 18:6 hours light: dark cycle. The growth medium was replaced weekly. Preliminary experiments with L. minor plants exposed to 50 µM menadione were performed to confirm that the imposed stress was not lethal to the plants.

2.2 Marker loading and exposure to the tested chemicals 10 mM H2 DCFDA (Calbiochem) stock solution was prepared in ethanol. L. minor plants used to visualize symplastic ROS formation were loaded with H2 DCFDA by immersing whole plants in modified Steinberg growth medium containing 50 µM H2 DCFDA. After two hours of loading, the plants were rinsed for 5 minutes in fresh growth medium to remove extracellular H2 DCFDA. L. minor plants used to visualize apoplastic ROS formation were not pre-loaded with the marker - H2 DCFDA was added to the growth medium (final concentration of 50 µM) in the test vessels and was not removed prior to the exposure of the plants to the tested chemicals. 10 mM menadione and 10 mM ascorbate (both from Sigma) were prepared in ethanol or distilled water, respectively. Whole L. minor plants were exposed to menadione (50 µM), menadione (50 µM) + ascorbate (100 µM) or neither for control by adding the appropriate quantities of the chemicals to the test vessels with the growth medium.

2.3 Measuring apparatus set-up Immediately after the addition of the chemicals to the test vessels, the vessels with the exposed plants were transferred to the measuring chamber where they remained throughout the experiment. The plants were continuously illuminated (15 µ mol m−2 s−1 PAR) with a 450 nm high power LED (LED-450-01U, Roithner Lasertechnik, Austria) with a band pass filter 447 ± 30 nm (Semrock FF01-447/60-25, USA). An illumination ring was used to ensure optimal and homogenous illumination. Images were captured by an EMCCD camera (L3Vision CCD65, e2v Technologies, UK) equipped with a band pass filter 536 ± 20 nm (Semrock FF01-536/40-25, USA) using Movie box deluxe frame grabber and Pinnacle Studio Plus V9.42 software (both from Pinnacle Systems, USA). EMCCD

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is a quantitative digital camera technology that is capable of detecting ultra-weak light whilst maintaining high quantum efficiency, large dynamic range, and fast frame rates, achievable by way of the electron multiplying structure built into the sensor.

2.4 Data acquisition and extraction One second digital video clips were captured with a frame rate of 24 images per second at each designated incubation time (0, 30, 60, 120, 180, 240, 300 and 360 min). Individual digital images were extracted from the video clips and the first 10 averaged to reduce noise using custom made software. The obtained averaged digital images were then specifically analyzed according to the H2 DCFDA loading protocol. In the apoplastic ROS generation studies, the fluorescent signal from the medium was analyzed whereas in the symplastic studies the fluorescent signal from individual L. minor fronds was analyzed. Four areas (109 pixels each) of the fluorescing medium per treatment were measured in the apoplastic studies, whereas one area per individual frond was measured in the symplastic studies (all fronds were analyzed, not only the fluorescing ones). The result was calculated as the average grey value of the selected areas. The same areas were followed for analysis at each time interval.

2.5 Statistical analysis All data were analyzed by computer software MS Excel 2003 and GraphPad Prism 4 for Windows. Two-way analysis of variance and Bonferroni post tests were performed on fluorescence data. The number of experiment repetitions, replicates and statistical significance is indicated in the figure captions.

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Results

3.1 Method validation Several experiments were performed prior to the main experiments to ensure method validity. We estimated the effect of AAPH, a peroxyl radical generator, and H2 O2 on fluorescence intensity accumulation. Both chemicals (final concentration of 1 mM) were added to the test vesicles containing growth medium with 100 µM H2 DCFDA and L. minor plants. Fluorescence intensity accumulation was measured for 15 minutes and the slopes of individual fluorescence curves calculated. The slope of control, AAPH, or H2 O2 treated plants were 4.7 ± 0.7, 10.9 ± 1.3 and 16.1 ± 2.1 A.U. min−1 , respectively. We also tested the effect of various concentrations of H2 DCFDA on apoplastic fluorescence intensity accumulation in control plants. The calculated slope of fluorescence curve was 3.1-fold greater in growth medium containing the test plants and 100 µM H2 DCFDA compared to 50 µM H2 DCFDA. Due to relatively fast saturation of the fluorescence signal in the case of 100 µM H2 DCFDA, we used 50 µM H2 DCFDA in the main experiments. Additionally,


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we tested the effect of various concentrations of menadione (1, 5 and 50 µM) on apoplastic fluorescence intensity accumulation. We calculated maximal slopes of individual fluorescence curves from apoplastic fluorescence data and normalized them to control. 50 µM menadione caused the biggest increase in fluorescence intensity - 3.07-fold greater than control. Fluorescence intensity accumulation in 1 and 5 µM menadione treated plants was only 1.03- and 1.17-fold greater than control, respectively. Therefore, we used 50 µM menadione in the main experiments. We have also tested the effect of L. minor rinsing on esterase activity. We have put rinsed or non-rinsed L. minor into test vesicles containing growth medium with 50 µM H2 DCDA. After 3 hours we measured the fluorescence intensity of the medium. The rinsed L. minor exhibited only slightly smaller fluorescence intensity (9.1%) and the difference was not significantly different from non-rinsed L. minor.

3.2 Visualization of ROS generation Throughout the experiment, the test vessels with whole, intact L. minor plants were in the measuring chamber. Therefore, besides fronds, roots can be seen in some images. A digital colour photograph of a typical test vessel set-up can be seen in Figure 1. Several controls were included in the experiment. No fluorescence increase was observed in test vessels 1, 3, 4, 6, 7 and 9, therefore only the test vessels 2, 5 and 8 were further analyzed (for details of exposure conditions, see Figure 1). In the apoplastic ROS visualization experiments, where H2 DCFDA was present throughout the experiment in the test medium, we observed an increase of fluorescence in the test medium and L. minor roots (Figure 2a). In the symplastic ROS visualization studies, where L. minor plants were preloaded with H2 DCFDA and the marker was rinsed from the loaded fronds before the experiment, we observed a strong increase of fluorescence in some L. minor fronds exposed to 50 µM menadione (Figure 2b) and also an increase of fluorescence of the medium in the case of 50 µM menadione treated L. minor (observe the increase of medium fluorescence in Figure 2b).

3.3 Apoplastic ROS generation The strongest fluorescence in the test medium was detected in the test vessels of 50 µM menadione treated L. minor plants (Figure 2a). The signal increased less over time in menadione (50 µM) + ascorbate (100 µM) treated plants and the least in the control plants. Also, strong and fast fluorescence increase was observed in the roots of 50 µM menadione treated L. minor plants. Menadione caused a statistically significant increase in fluorescence of the test medium containing H2 DCFDA after 60 minutes of exposure compared to control and after 120 min compared to both menadione and ascorbate treated L. minor plants (Figure 3). The addition of ascorbate (100 µM) to the test medium decreased the fluorescence intensity accumulation so that significant increase compared to control values was noticed only after

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240 min. We calculated maximal slopes of individual fluorescence curves from apoplastic fluorescence data and normalized them to control. Menadione caused the biggest increase in fluorescence intensity, indicated by the largest slope (3.11±0.11 A.U. min−1). Both menadione alone and the mixture of both menadione and ascorbate(1.53±0.11 A.U. min−1) caused the fluorescence intensity to increase significantly (p < 0.0001) faster compared to control (1.00 ± 0.03 A.U. min−1 ).

Fig. 1 Digital colour photograph of a typical test vessel set-up containing whole L. minor plants. In the bottom middle and right test vessel, the roots can be seen. All vessels contain L. minor growth medium. The vessels additionally contain: 1 − 50 µM H2 DCFDA, 2 - L. minor +50 µM H2 DCFDA +50 µM menadione, 3 - L. minor +50 µM menadione, 4 - L. minor, 5 - L. minor +50 µM H2 DCFDA +50 µM menadione +100 µM ascorbate, 6 - L. minor +50 µM menadione +100 µM ascorbate, 7 - L. minor, 8 - L. minor + 50 µM H2 DCFDA, 9 - L. minor.

3.4 Symplastic ROS generation We observed a strong increase of fluorescence intensity accumulation in 57% of L. minor fronds which were pre-loaded with H2 DCFDA and exposed to 50 µM menadione(Figure 2b). Since we analyzed all pre-loaded fronds to remain impartial, this increase of symplastic fluorescence intensity accumulation of menadione treated plants was not significantly different from control (Figure 4). The exposure of L. minor plants to both menadione and ascorbate lowered the symplastic fluorescence increase to control levels. After 120 min we


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Fig. 2 Averaged greyscale digital fluorescent images of Lemna minor in test vessels (a) with growth medium containing 50 µM H2 DCFDA for apoplastic ROS visualization or (b) without H2 DCFDA in the growth medium for symplastic ROS visualization. In (b) Lemna minor plants were pre-loaded with H2 DCFDA for 2 hours prior to exposure to the tested chemicals. At time 0, L. minor plants were exposed to 50 µM menadione (M), 50 µM menadione +100 µM ascorbate (M + A) or neither for control (C). In the apoplastic studies (a) the measurement areas are indicated by the black circles whereas in the symplastic studies (b) the measuring areas are indicated by the white circles. The same areas were measured at all designated times. measured an insignificant decrease in the average fluorescence in all treatments (Figure 4). The fluorescing dye was not entirely retained in the symplast but instead, flowed outside the plant tissue into the surrounding medium in which we observed a 2.2-fold increase of fluorescence in the case of 50 µM menadione treated L. minor (observe Figure 2b).

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Fig. 3 Apoplastic ROS visualization - increase of Lemna minor growth medium fluorescence over time. L. minor plants were kept in test vessels with growth medium containing 50 µM H2 DCFDA. At time 0, L. minor plants were exposed to 50 µM menadione (M), 50 µM menadione + 100 µM ascorbate (M + A) or neither for control (C). Four areas of fluorescing growth medium per treatment were measured as indicated in Figure 2a. The results represent means ± SE from two independent experiments (n = 8). Statistical significance is represented by letters: a - significantly different from controls, b - significantly different from M + A (p < 0.01). A.U. - arbitrary units. We calculated the slopes of individual symplastic fluorescence curves in the interval from 0 to 60 min and normalized them to control. Menadione alone caused the largest increase of fluorescence intensity in 57% of fronds indicated by the biggest average slope (4.97 ± 1.79 A.U. min−1 ). In L. minor plants exposed to both menadione and ascorbate (1.30 ± 0.39 A.U. min−1 ), symplastic fluorescence increase was not significantly higher than control (1.00 ± 0.23 A.U. min−1 ).

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Discussion

We have constructed an experimental set-up which allowed us to visualize ROS generation non-invasively in real-time, on a whole plant level. Most of the plant ROS visualization studies were otherwise performed on cell cultures [7, 9]. The main drawback of these studies is dubious extrapolation of results to intact higher plants where the cells originated from. Several authors reported ROS visualization in higher plants [6, 23] but using very invasive plant tissue processing techniques. We were primarily interested in visualizing ROS non-invasively and therefore did not process the test plants. The main advantage of using an ultra-sensitive EMCCD camera was that we did not influence the experimental system. Measuring light intensity of 15 µM m−2 s−1 PAR was much lower than the light intensity under which the plants were grown (160 µM m−2 s−1 PAR), which indicates that nonphotochemical fluorescence quenching was not induced. Thus the low measuring


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illumination did not influence the photosynthesis significantly but at the same time provided enough excitation light to obtain a good measuring signal. Additionally, the low excitation light did not quench dichlorofluorescein significantly (Figure 2, Figure 4) as is often the case when this dye is used in fluorescence microscopy [24]. The decrease in symplastic fluorescence after 120 min (Figure 4) could be attributed to a flux of the dye from the symplast to the surrounding medium [11].

Fig. 4 Symplastic ROS visualization - increase of Lemna minor fronds’ fluorescence over time. L. minor plants were pre-loaded with H2 DCFDA for 2 hours, then transferred to test vessels with fresh growth medium prior to exposure to the tested chemicals. L. minor plants were exposed to 50 µM menadione (M), 50 µM menadione + 100 µM ascorbate (M + A) or neither for control (C). The results represent digital fluorescence signal originating from the fronds as indicated in Figure 2b. All fronds were measured, to insure impartiality of the results. Values presented are means ± SE from a single experiment (n = number of fronds marked by white circles in Figure 2b). A.U. - arbitrary units. The metabolism of menadione creates substantial quantities of superoxide and secondarily, via enzymatic or spontaneous dismutation, hydrogen peroxide in isolated hepatocytes [17]. It was also used as a ROS inducer in yeast cells [8], fruits of Capsicum annuum [18], glial cells [19], Pisum sativum [20], Arabidopsis thaliana cell culture [21] and Arabidopsis thaliana plants [22]. Ascorbate, on the other hand, is - besides glutathione - the plants’ main low molecular weight antioxidant and can directly reduce superoxide as well as hydrogen peroxide [25]. We measured a significant reduction of fluorescence intensity accumulation in the apoplastic experiments (Figure 3) when ascorbate was added indicating its quenching of ROS generated by menadione. Plant cells have a substantial extracellular antioxidative potential fuelled by the action of cell-wall peroxidases which use apoplastic antioxidants, mainly ascorbate and phenolic substances, as reducing agents [26]. Extracellular antioxidants, unlike intracellular ones, which are continually replenished by reserves of NADH or NADPH, have to be transported into the cell to be recycled; this process takes time [26]. By adding ascorbate to the test

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medium we have thus effectively reduced the magnitude of oxidative stress induced by menadione. The number of fronds or frond area did not exhibit a major influence on H2 DCFDA fluorescence when compared to the effect of menadione (Figure 2). The resulting fluorescence observed in the test medium containing H2 DCFDA was the result of the combined action of plant apoplastic esterases and ROS (Figure 2a, Figure 3). The apoplastic esterases cleaved the H2 DCFDA present in the medium to H2 DCF. Nonfluorescent H2 DCF was then oxidized to fluorescent dichlorofluorescein (DCF) by ROS generated by the apoplast of the stressed plants. Since H2 DCFDA must be hydrolyzed by the action of unspecific esterases to non-fluorescent H2 DCF which can be oxidized to fluorescent DCF [10], esterases must be present and active. Therefore, it could be argued that H2 DCF would be a better marker of apoplastic oxidative stress since it would not need unspecific esterases to become oxidized. However, it is known that pectin methyl esterases are ubiquitous enzymes in plants [14], present in the cell wall. They are necessary for cell wall reassembly and degradation [15] and their transcripts were found in all plant tissues [16]. Less is known about their specificity, but according to our results, it is evident that esterases which hydroxylize H2 DCFDA to H2 DCF are abundant in the apoplast (Figure 2a). A similar increase of H2 DCFDA fluorescence in the medium where Lemna gibba was present was reported by Kandeler (1993) [13]. Additionally, the investigated organism or cells must not be damaged to such an extent that the unspecific esterases would be inactivated. In our preliminary studies, we observed little or no fluorescence from fronds and the complete lack of fluorescence from the roots when L. minor plants were exposed to 500 µM menadione (not shown). This was probably due to too intense toxicity of the tested menadione concentration which resulted in the death of root cells and consequent esterase inactivation. Therefore, prior to the main experiments, we determined the concentration of menadione that initiated strong fluorescence increase, but was not lethal to the plant cells (observe the fluorescing roots in Figure 2a). Thus, the ester form of fluorescein (H2 DCFDA) is a more appropriate choice for apoplastic ROS detection since it also tests for too acute stress which would otherwise result in false negative results. We observed high variation of symplastic fluorescence (Figure 2b and Figure 4), probably due to differences in H2 DCF uptake. Kandeler (1993) [13] proposed that this uptake is an active process, mediated by carriers of gibberelic acid-like substances. The author reported high fluorescent signals in Lemna gibba plant tissue after 5 − 6 hours of dyeloading. In itself this would not pose a problem as we would simply extend the loading period. However, in the preliminary experiments we discovered that after long loading periods (16 h), most of the H2 DCF was already oxidized to fluorescent DCF prior to the addition of the investigated chemicals. Thus, we measured no increase in the fluorescence of 16-hour pre-loaded L. minor fronds after exposing them to menadione. This is probably due to normal metabolic processes which always generate ROS [25] combined with the action of endogenous peroxidases, which can induce H2 DCF oxidation without the presence of ROS [2] - note the slow but constant fluorescence increase in control L. minor plants (Figure 2). Additionally, we observed that the dye was not entirely retained in


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the symplast but instead, flowed outside the plant tissue into the surrounding medium in which we observed a 2.2-fold increase of fluorescence in the case of 50 µM menadione treated L. minor (observe Figure 2b). Therefore, one could consider using the more expensive, carboxylated, form of H2 DCFDA (carboxy-H2 DCFDA) because of its better membrane permeability and better retention in the intracellular space [11]. According to our results, it could be argued that yeast cells [8] or plant cell cultures [7, 9] are probably better test subjects than L. minor plants for intracellular ROS generation studies. This is primarily because of their big surface-to-volume ratio that enables faster dye uptake and thus short loading periods. In such assays, the loading medium is removed after a relatively brief period of loading (10 − 20 min), the cells are rinsed to remove the non-absorbed dye, homogenized and centrifuged. Fluorescence is then measured in the supernatant and thus intracellular oxidative stress is estimated. The loading times needed to load L. minor plants with adequate quantities of H2 DCF proved to be too long to ensure good experimental reproducibility in symplastic studies.

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Conclusions

We have constructed an experimental set-up which enabled us to visualize menadioneinduced ROS formation in vivo on a whole plant level, non-invasively. The high sensitivity of the measuring system allowed us to use a weak excitation light which did not induce significant photobleaching of the fluorescing probe. We have observed that the addition of ascorbate to the test medium containing menadione decreased apoplastic and symplastic oxidative stress in L. minor plants. The assay is quick and straightforward and can be applied to a range of chemicals in various physiological and toxicological plant studies.

Acknowledgements The research was financed by the Slovenian Research Agency, grants Nr. 3311-04-831833, J1-6473, L4-6222, V4-0106 and L1-5146. We are grateful to Dr. Maja Berden Zrimec for help with sustaining a healthy duckweed culture and Dr. Kristina Sepˇcić for valuable discussions.

References [1] R. Kohen and A. Nyska: “Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification”, Toxicol. Pathol., Vol. 30, (2002), pp. 620–650. [2] M.M. Tarpey and I. Fridovich: “Methods of detection of vascular reactive species nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite”, Circ. Res., Vol. 89, (2001), pp. 224–236.

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[3] M. Grant, I. Brown, S. Adams, M. Knight, A. Ainslie and J. Mansfield: “The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death”, Plant J., Vol. 23, (2000), pp. 441–450. [4] M.J. Fryer, K. Oxborough, P.M. Mullineaux and N.R. Baker: “Imaging of photooxidative stress responses in leaves”, J. Exp. Bot., Vol. 53, (2002), pp. 1249–1254. [5] C. Ortega-Villasante, R. Rellan-Alvarez, F.F. Del Campo, R.O. Carpena-Ruiz and L.E. Hernández: “Cellular damage induced by cadmium and mercury in Medicago sativa”, J. Exp. Bot., Vol. 56, (2005), pp. 2239–2251. [6] K.S Gould, J. McKelvie and K.R. Markham: “Do anthocyanins function as antioxidants in leaves? Imaging of H2 O2 in red and green leaves after mechanical injury”, Plant. Cell Environ., Vol. 25, (2002), pp. 1261–1269. ´ Sárdi, [7] Z. Bozsó, P.G. Ott, A. Szatmari, A. Czelleng, G. Varga, E. Besenyei1, E. ´ Bányai and Z. Klement: “Early detection of bacterium-induced basal resistance E. in tobacco leaves with diaminobenzidine and dichlorofluorescein diacetate”, J. Phytopathol., Vol. 153, (2005), pp. 596–607. [8] W. Jakubowski and G. Bartosz: “Estimation of oxidative stress in Saccharomyces cerevisae with fluorescent probes”, Int. J. Biochem. Cell. B., Vol. 29, (1997), pp. 1297–1301. [9] I.B. Gerber and I.A. Dubery: “Fluorescence microplate assay for the detection of oxidative burst products in tobacco cell suspensions using 2’,7’-dichlorofluorescein”, Methods in Cell Sci., Vol. 00, (2003), pp. 1–8. [10] J.-H. Yang, S.F. Basinger, R.L. Gross and S.M. Wu: “Blue light-induced generation of reactive oxygen species in photoreceptor ellipsoids requires mitochondrial electron transport”, Invest. Ophth. Vis. Sci., Vol. 44, (2003), pp. 1312–1319. [11] R.P. Haugland: “Handbook of fluorescent probes and research products, 9th edition”, Molecular probes Inc., Oregon, 2002, pp. 966. [12] W. Wang: “Literature review on duckweed toxicity testing”, Environ. Res., Vol. 52, (1990), pp. 7–22. [13] R. Kandeler: “Fluorescein accumulation in flower primordia”, J. Plant. Physiol., Vol. 142, (1993), pp. 695–698. [14] J. Caffe, M.E. Tiznado and A.K. Handa: “Characterization and functional expression of a ubiquitously expressed tomato pectin methylesterase”, Plant Physiol., Vol. 11, (1997), pp. 1547–1556. [15] T. Girke, J. Lauricha, H. Tran, K. Keegstra and N. Raikhe: “The cell wall navigator database. A systems-based approach to organism-unrestricted mining of protein families involved in cell wall metabolism”, Plant Physiol., Vol. 136, (2004), pp. 3003– 3008.


363

[16] J. Geisler-Lee, M. Geisler, P.M. Coutinho, B. Segerman, N. Nishikubo, J. Takahashi, H. Aspeborg, S. Djerbi, E. Master, S. Andersson-Gunneras, B. Sundberg, S. Karpinski, T.T. Teeri, L.A. Kleczkowski, B. Henrissat and E.J. Mellerowicz: “Poplar carbohydrate-active enzymes. Gene identification and expression analyses”, Plant Physiol., Vol. 140, (2006), pp. 946–962. [17] H. Thor, M.T. Smith, P. Hartzell, G. Bellomo, S.A. Jewell and S. Orrenius: “The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes”, J. Biol. Chem., Vol. 257, (1982), pp. 12419–12425. [18] F. Bouvier, R.A. Backhaus and B. Camara: “Induction and control of chromoplastspecific carotenoid genes by oxidative stress”, J. Biol. Chem., Vol. 273, (1998), pp. 30651–30659. [19] S.B. Hollensworth, C.-C. Shen, J.E. Sim, D.R. Spitz, G.L. Wilson and S.P. Ledoux: “Glial cell type-specific responses to menadione-induced oxidative stress”, Free Radical Bio. Med., Vol. 28, (2000), pp. 1161–1174. [20] V.D. Samuilov, E.M. Lagunova, E.V. Dzyubinskaya, D.S. Izyumov, D.B. Kiselevsky and Y.V. Makarova: “Involvement of chloroplasts in the programmed death of plant cells”, Biochemistry (Mosc.), Vol. 67 (2002), pp. 627–634. [21] A.J. Meyer and M.D. Fricker: “Control of demand-driven biosynthesis of glutathione in green Arabidopsis suspension culture cells”, Plant Physiol., Vol. 130, (2002), pp. 1927–1937. [22] K. Yoshinaga, S. Arimura, Y. Niwa, N. Tsutsumi, H. Uchimiya and M. KawaiYamada: “Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana”, Ann. Bot. (Lond.), Vol. 96, (2005), pp. 337–342. [23] M.E. Maffei, A. Mithöfer, G.-I. Arimura, H. Uchtenhagen, S. Bossi, C.M. Bertea, L.S. Cucuzza, M. Novero, V. Volpe, S. Quadro and W. Boland: “Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide”, Plant Physiol., Vol. 140, (2006), pp. 1022–1035. [24] L. Song, C.A. Varma, J.W. Verhoeven and H.J. Tanke: “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy”, Biophys. J., Vol. 70, (1996), pp. 2959–2968. [25] G. Noctor and C.H. Foyer: “Ascorbate and glutathione: Keeping active oxygen under control”, Annu. Rev. Plant Phys., Vol. 49, (1998), pp. 249–279. [26] C.J. Baker and N.M. Mock: “A method to detect oxidative stress by monitoring changes in the extracellular antioxidant capacity in plant suspension cells”, Physiol. Mol. Plant P., Vol. 64, (2004), pp. 255–261.